BRPI0621953B1 - solid, dry composition, effective in the biological control of colletotrichum gloeosporioides; method for producing a composition; method for biological control of colletotrichum gloeosporioides disease and method for reducing weight loss during mango storage - Google Patents

Abstract

Method for the production of a solid and dry composition, solid, dry composition effective in the biological control of colletotrichum gloeosporioides, method for the biological control of disease caused by colletotrichum gloeosporioides and method for reducing weight loss during storage of mango. The present invention describes a method for producing a solid, dry and biologically effective composition of coiletotrichum gloeosporioides which comprises rhodotorula minuta and has a shelf life of less than one year. It also describes the resulting composition and a method for its use as a biological control agent. Likewise, the present invention discloses a solid and dry composition comprising rhodotorula minuta with a shelf life of up to one year under refrigeration and a second biological control agent, bacillus subtilis, with which control levels of the shelf life are obtained. severity of anthracnosis equal to or better than those obtained at higher doses than those used with these same control agents when applied independently. The present invention also also describes a method for reducing weight loss during storage of postharvest mango.

Description

SOLID, DRY, EFFICIENT COMPOSITION ON THE BIOLOGICAL CONTROL OF COLLETOTRICHUM GLOEOSPORIOIDS; Method for the production of a composition; METHOD FOR BIOLOGICAL CONTROL OF NURSES CAUSED BY COLLETOTRICHUM GLOEOSPORIOIDS AND METHOD TO REDUCE WEIGHT LOSS DURING MANGO STORAGE

TECHNICAL FIELD The present invention relates to methods for obtaining solid compositions with Rhodotorula minuta, an effective biological control agent against Colletotrichum gloeosporioides, which causes the main fungal disease of mango. Said methods allow obtaining an effective biofungicide with a long shelf life. These compositions may comprise, in addition to Rhodotorula minuta, another biological control agent such as Bacillus subtilis, to reduce the dose of control agents. It also refers to the pre-harvest application of such compositions for the biological control of the Colletotrichum gloeosporioides phytopathogen mushroom without negatively affecting the main parameters of fruit quality. Similarly, the present invention also relates to a method for reducing weight loss during storage of mango, characterized in that at least one application of a dry solid composition with Rhodotorula minuta is applied to the pre-harvest. effective for the biological control of Colletotrichum gloeosporioides.

BACKGROUND OF THE INVENTION

Mango fungal diseases, such as anthracnose, can reduce fruit quality and cause post-harvest losses of up to 60% of total production (Vega, 2001). In extreme cases, a high incidence of this disease can cause losses of up to 100% of fruits produced in climates with high humidity (Arauz, 2000). This disease is caused by the phytopathogenic mushroom Colletotrichum gloeosporioides Penz (Freeman et al., 1998). Mango anthracnose has a very high incidence in virtually all fruit producing areas. The pathogenic mushroom usually infects the mango tree from the early stages of the production cycle. As a result, the disease should be controlled primarily at preharvest as it affects tree productivity and although symptoms may not be evident at the time of cutting the fruit, they will manifest with the course of pest management. -harvest. Although many producers carry out postharvest chemical fungicide treatments, the preharvest control of anthracnose is crucial in the final quality of the fruits (Arauz, 2000). Biological control of plant diseases is a viable alternative to chemical control of plant pathogens. This implies the use of microorganisms (usually from the same pathogen habitat) that inhibit or impede the development of the pathogenic microorganism. These microorganisms have a high specificity and environmental safety (Spadaro and Gullino, 2004). Among biological antagonists stand out bacteria and yeast, the latter because they generally tolerate many of the chemical fungicides used in pre and / or postharvest (Spadaro and Gullino, 2004), allowing growers to use them in combination with fungicides. low toxicity chemicals.

Rhodotorula yeasts (particularly Rhodotorula glutinis) have been reported to be good biological control agents (Shanmuganathan, 1996; Chand-Goyal and Spotts, 1998) against Penicillum expansum in apples and pears (Benbow and Sugar, 1999) and against Botrytis. strawberry cinerea when applied before and / or after harvest (Helbig, 2001). Rhodotorula species are generally considered safe for humans. Naidu et al. (1999) demonstrated that oral feeding of frozen cells, between 0.5 and 6.0 g / kg body weight, of this yeast did not produce toxic effects in male and female albino rats. Specifically for Rhodotorula minuta, the American Type Culture Collection (AT CC, www.atcc.orq) has ranked it at Biosafety Level 1, meaning that it is not recognized as a cause of diseases affecting the health of adult humans. Biological control of fungal diseases has been reported, among other fruits, in apples, avocados, papayas and mangoes. For diseases caused by Penicilllium spp., Botrytis spp. Mucor spp., Pezicula spp., Phialophora and / or Monilinea spp., In apples it is recommended to use mixtures of: Cryptococcus infirmo-miniatus, Cryptococcus laurentii, Rhodotorula aurantiaca and Rhodotorula glutinis (Chand-Goyal and Spotts, 1998). For diseases caused by Pseudocercospora purpurea, in avocados it is recommended to use Bacillus subtilis (Korsten et al., 1997). For diseases caused by C. gloeosporioides, the use of Candida oleophila in papaya (Gamagae et al., 2004) is recommended for papaya and De Jager et al. For mango anthracnose. (2001) investigated the application of Bacillus spp. , discovering that latent infections could be controlled. Another successful case for mango was the postharvest application of Pseudomonas fluorences, reported by Koomen and Jeffries (1993). However, in none of the previous cases was evidence reported at the semi-commercial or commercial level. Only the authors of the present invention have reported Rhodotorula minuta yeast as a biological control agent.

Other authors have reported producing Rhodotorula minuta for other purposes, for example to produce and extract pigments (beta carotenes). Velankar and Heble (2003) reported that R. minuta can grow at pH values between 4 and 9 and at temperatures between 20 and 30 ° C, with cultivation in different liquid media.

Carrillo-Fasio et al. (2005) reported Bacillus subtilis and Rhodotorula minuta yeast as effective biological control agents against mango anthracnose caused by the phytopathogen mushroom Colletotrichum gloeosporioides Penz. The bacterium presented the phenomenon of antagonism by antibiosis and it was determined that Rhodotorula minuta yeast presented mainly the competence phenomenon (Patino-Vera et al., 2005). Bacillus subtilis and Rhodotorula minuta strains are available from internationally recognized microorganism deposits.

The authors of the present invention (Patino-Vera et al., 2005) reported the biological control of mango anthracnose in semi-commercial field trials at three production seasons (a requirement for corroborating efficacy) by applying liquid formulations. Rhodotorula minuta yeast pre-harvest. To produce sufficient viable yeast to carry out such semi-commercial applications, the submerged fermentation production process was successfully scaled from the laboratory test flask to a 100 L bioreactor employing a low culture medium. cost, being the first to report a scaling process to obtain viable R. minuta cells at high concentrations (up to 2 x 109 CFU / mL). The viable cells obtained in the pilot fermenter exceeded the values obtained in the shaken flasks and were obtained in a shorter time. Yeasts were harvested at the exponential phase of growth.

However, all the formulations tried so far presented a major technical problem: they lacked a shelf life with sufficient cooling for their marketing. Yeast viability drops rapidly within the first two months of storage. The minimum recommended shelf life is six months (Janisiewicz and Korsten, 2002), to be compatible with routine handling and storage practices in the agrochemical market. By formulating the yeast at a concentration of 1 x 109 CFU / mL and the addition of glycerol (20%) and xanthan gum (5 g / L), it was possible to avoid bacterial contamination and cell sedimentation. up to 107 CFU / mL for six months (Patino-Vera et al., 2005). This may be an insufficient cellular concentration, as doses of up to 108 CFU / mL of R. minuta may be required to achieve anthracnose control results similar to or better than those achieved when chemical fungicides such as Benomilo are used. The problem is aggravated because the loss of viability is increased as the cellular concentration of R. minuta increases. A liquid composition with an initial concentration of 101D CFU / mL R. minuta, and a phosphate pH buffer showed a dramatic drop in living cell concentration of up to five orders of magnitude after one month of refrigerated storage, compared to that initially formulated with 109 CFU / mL yeast (Patino-Vera et al., 2005).

As a result, research into alternatives to increase the shelf life of concentrated formulations was indispensable. In a practical sense, Janisiewicz and Jeffers (1997) pointed out that the major obstacle in the commercialization of products for biological control is the development of formulations with a stable shelf life, which allows maintaining an object disease severity control activity similar to that obtained. with products made with fresh cells, in whose formulations the vast majority of cells are alive.

To counteract the above, different strategies have been proposed, but they have not adequately solved the problem when the control agents are yeast or damp mushrooms. Among other alternatives, very costly processes (such as lyophilization) were employed to dry formulations of biological control agents. For example, Abadias et al. (2001) report that while employing protective agents such as skimmed milk powder, lactose, fructose, glucose, sucrose and / or various combinations thereof, as well as different means of rehydration, the efficacy of lyophilized cells from another yeast, Candida sake , was significantly smaller than cells without lyophilization. Cheaper processes such as spray drying have not been effective in preserving bacteria, mushroom conidia or yeast. For example, for spray-dried and refrigerated lactic bacteria, a considerable decrease in viability has been reported after three months of storage (Wan-Yin and Mark, 1995). Jones et al. (2004) reported that, in general, sprays dried biological control agent Coniothyrium minitans conidia had a lower germination percentage than those that were not dried. In the case of another biological control agent (Pantoea agglomerans), a recovery percentage of approximately 50% after spray drying and a low final viability are reported (Costa et al. 2002). In recently reported evidence, Abadias et al. (2005) indicated that with spray drying of Candida sake yeast, this biological control agent is significantly less effective against an apple phytopathogen mushroom than fresh cells. These authors conclude that spray drying is not a good method for yeast dehydration because few of them survive (only about 10%), poor product recovery is achieved and, above all, ineffective in controlling the disease.

The viability of drying Rhodotorula yeasts for other purposes has previously been reported, for example for the production and extraction of beta-carotenes employing Rhodotorula glutinis. Bhosale et al. (2003) reported that the culture broth with this yeast can be concentrated up to ten times by microfiltration and subsequently spray drying, resuspending the concentrated yeast at concentrations of 1 to 15% (w / v) at temperatures 40 to 200 ° C, and with outlet temperatures of 33 to 84 ° C. Detecting viable cells after the drying process at an inlet temperature range of 40 to 180 ° C and outlet temperature of 33 to 82 ° C.

Abadias et al. (2005) used skimmed milk powder, skimmed milk powder plus lactose or magnesium sulfate as protective carriers or carriers in the drying of the biological control agent Candida sake. On the other hand, Lodato et al. (1999) report using sugars and polymer matrices (maltodextrins) as a support in freeze drying. These same authors report that incorporation of carbohydrates into the suspension composition of microorganisms prior to drying helps to preserve their viability and increase shelf life of the dried product. This is possibly due to the ability of carbohydrates to establish hydrogen bridges that help stabilize vital proteins. They also report that the degree of protection of these carbohydrates is conferred by their low molecular weight saccharide content. The carbohydrates with the highest content of low molecular weight saccharides showed the highest degree of protection. On the other hand, the formulation of the biological control agent with supports or protectors increases the recovery of dry dust and helps to control its moisture content (Abadias et al., 2005).

Larena et al. (2003) demonstrated that, in addition to spray drying, it is possible to dry Penicillum oxalicum conidia by freeze drying or fluid bed drying. The use of these last two processes makes it possible to preserve 100% of their viability, but it is necessary to formulate previously with high cost protective supports, such as Tween 20 skimmed milk powder or more peptone milk. Without the addition of these supports or protectors, the conidia did not maintain their viability at room temperature, while with the addition of protectors they maintained a viability of 40 to 50% even for 180 days. With spray drying, the viability value fell to 20%. Finally, they proved that fluidized bed dried conidia retained their effectiveness as biological control agents.

For all of the above, it is indispensable to investigate alternatives for the formulation and packaging of biological control agents that allow their adequate conservation for adequate periods for their commercialization. On the other hand, several authors have reported inconsistent efficacy of biological control agents when they are applied to commercial orchards (Janisiewicz and Jeffers, 1997; Janisiewicz and Korsten, 2002; Guetsky et al., 2001). These inconsistencies and other benefits can be obtained by employing compatible mixtures of biological control agents (Janiciewicz and Jeffers, 1997; Janisiewicz and Korsten, 2002). Therefore, it is also necessary to have alternatives to compatible mixtures of biological control agents to overcome such inconsistencies and to obtain other benefits, such as reducing the doses of required antagonist microorganisms (Spadaro and Gullino, 2004) and thereby contributing to the fight against viable cell losses during formulation, concentration and / or drying processes.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Graph showing a growth kinetics and glucose consumption of Rhodotorula minuta yeast grown in a 10 L fermenter.

Figure 2. The graph illustrates the recovery efficiencies of CFU and total solids in Rhodotorula minuta spray drying processes of centrifuged and non-centrifuged fermentation broths.

Figure 3. Comparison of a "control kinetics" of percent germination of C. gloeosporioides, without the presence of R. minuta and an example in which a previously formulated solid and dry composition with R. minuta is added.

Figure 4. The graph shows a comparison of the antagonistic effect of Rhodotorula minuta liquid and solid formulations, measured as the percentage of conidia germination, at 56 hours of co-cultivation.

Figure 5. Graph showing the concentration of viable R. minuta cells in the solid and liquid shelf-life formulation.

Figure 6. This graph shows the behavior of viable R. minuta cells during an accelerated aging treatment (37 ° C storage) of a supported R. minuta formulation and a control.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based on prior investigations by its authors which have made them the first in the world to report Rhodotorula minuta yeast as a biological control agent against mango anthracnosis (Patino- Vera et al., 2005) and the various substantial technical improvements that have been incorporated into the production process, formulation, drying and application of biological control agents (independently or in combination) against C. gloeosporioides, causing anthracnose, particularly in mango. Said improvements allowed them to establish the method of the present invention comprising a first step consisting of a Rhodotorula minuta cell production process; an optional second step of cellular packet recovery and resuspension; an optional third formulation step; and a fourth drying step to obtain a solid Rhodotorula minuta composition effective for the biological control of anthracnose caused by C. gloeosporioi des.

The method of the present invention provides a dry solid composition effective as a biological control agent and with extended shelf life under refrigeration (more than six months as recommended by Janisiewicz and Korsten, 2002). The invention also relates to a composition comprising R. minuta and a second effective biological control agent against C. gloeosporioides, particularly Bacillus suhtilis. The advantage of this composition is that it is effective in controlling the severity of anthracnose at doses lower than those required for these same control agents when applied independently. The invention also includes a method for the biological control of C. gloeosporioides comprising at least one pre-harvest application of effective doses of an R. minuta dry solid composition. The invention also includes a method for reducing weight loss during mango storage, characterized in that it is pre-harvested with at least one application of an R. minuta dry solid composition effective for biological control of C gloeosporioids.

The methods of the present invention are useful for dry compositions comprising R. minuta with a long shelf life and effective for controlling C. gloeosporioides, mango pathogen and other fruits, with an industrially scalable technology that employs raw materials. low cost, which allows the use of antagonist microorganisms individually or in combinations. The latter form makes it possible to reduce the dose of biological control agents required to control disease severity caused by the pathogenic mushroom. The biological products developed are easy to handle and apply, and have a shelf life of at least one year under refrigeration, which is very convenient and suitable for their eventual distribution and marketing in the agrochemical market.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS: The term "cultivar" refers to the propagation of organisms on or in cultivation media of various types, solid and liquid.

As used herein, "biological control of C. gloeosporioides" is defined as the act of decreasing, reducing, mitigating, stabilizing, reversing, slowing down or delaying the severity of disease caused by the C. gloeosporioides phytopathogen mushroom by application. one or more antagonistic microorganisms (biological control agents).

In the present invention the terms antagonist or biological control agent are used interchangeably to denote a microorganism with biological control activity of C. gloeosporioides.

An "effective product" is a product that, when applied in a sufficient amount, obtains biological control effects of C. gloeosporioides. An effective product may be administered in one or several applications. In terms of treatment and protection, an "effective treatment" is the dose and number of applications sufficient to obtain a biological control effect of C. gloeosporioides. Tree, as defined herein, includes most of the plant encompassing the buds, the stem, the knots, the inter-nodes, the petiole, the leaves, the flowers, the fruits and the like. The present invention solves the technical problem of the low shelf life of the previously reported R. minuta liquid compositions by a method for producing a solid and dry composition effective in the biological control of Colletotrichum gloeosporioides which comprises Rhodotorula minuta and has a shelf life of at least one year. It has been previously reported the production of Rhodotorula yeasts for their application in the biological control of plant diseases through submerged fermentation processes. However, most of these processes are carried out in test flasks or laboratory fermenters.

In addition, the reported processes refer to obtaining viable cells in full growth (exponential phase), using bacteriological or laboratory grade culture media. In order to corroborate the efficacy of field biological control agents, semi-commercial quantities of said agents are required.

For this, it is necessary to scale production processes at least to the Pilot Plant level, using low cost cultivation media. There are no reports from other authors specifically addressing the aspects of the Rhodotorula minuta production process to obtain cells resistant to the drying process, in which live cell recovery yields (measured as CFU / mL) are sufficient to formulate prototypes. semi-commercial products which, once dry, retain the effectiveness of controlling the severity of a fungal disease. In these last two aspects, the authors of the present invention were the first to obtain them, since, as already reported above, several authors have failed to develop dry products with high concentrations of effective biological control agents (Wan- Yin and Mark, 1995; Jones et al., 2004) including other yeasts used as biological control agents, which retain their effectiveness in the control of fungal diseases (Abadias et al., 2005).

Thus, the method for obtaining an effective composition comprising Rhodotorula minuta for the biological control of C. gloeosporioides consists of the following steps: production of adequate amounts of biomass, optionally cell package recovery and resuspension, optionally formulation by the addition of a support and drying of said formulations.

First stage: biomass production, the R. minuta production process is started by developing the inoculum from petri dishes, passing through agitated 2.8 L test flasks whose contents are transferred to a nursery bioreactor to get the seed cultivation. The pH is adjusted to an initial value of 4 to 9, a stirring of 100 to 300 rpm is performed to obtain good mass and heat transfer; The appropriate growth temperature is controlled, for example, from 20 to 30 ° C, with sufficient aeration within the first four hours, preferably 0.5 Air Volume per Medium Volume per Minute (WM) and increasing this by 50. and 100% of the initial value for the following sixteen hours of cultivation. At the end of this time, the seed cultivation is transferred to the production bioreactor containing a yeast extract enriched mineral medium with an initial pH between 4 and 9. Stirring between 100 and 400 rpm is performed to obtain a good transfer of mass and heat; The temperature suitable for growth is controlled, for example from 20 to 30 ° C, with sufficient aeration for proper growth, preferably 1 WM. Cultivation time may vary depending on the cultivation parameters used, but may vary from 40 to 65 hours. This time is the time required for R. minuta yeast to be in a stationary phase (see example 1), because several authors (Werner-Washburne et al., 1993; Panek and Panek, 1990; Plesset et al., 1987 ) report that another type of yeast, for example Saccharomyces cerevisiae, being in the stationary phase, is more resistant to several stress classes. After this time viable cells of R. minuta of the order of 1 x 10 9 CFU / mL are obtained.

Second step (optional): Cellular package recovery and resuspension. It consists in recovering the cellular package of Rhodotorula minuta yeast in order to eliminate the largest amount of water from the culture medium. For this it is possible to use unitary centrifugation or microfiltration operations (Bhosale et al., 2003). For centrifugation, for example, a Sharples-type tubular centrifuge may be employed. In this case it will be necessary to resuspend the cell package in a neutral pH buffer solution, such as phosphate, to obtain a concentrated suspension of Rhodotorula minuta yeast.

Third step (optional): formulation. It consists in the formulation of the biological control agent R. minuta, by the addition of a protective or carrier support, and various supports or carriers can be used among them those that are based on a high starch flour such as maltodextrins, flour. corn or cornstarch (see example 7).

In the present invention, a solid and dry composition of R. minuta cells can be obtained by adjusting the suspension to have a concentration of 3 to 20% solids (see example 2). This suspension feeds a dryer.

Fourth step: drying. This step may be performed, for example, in a spray dryer at an inlet temperature between 100 and 160 ° C and an outlet temperature between 50 and 70 ° C. Biomass concentrations between 1 and 15%, preferably 10% may be used; inlet temperatures between 40 and 180 ° C and an outlet temperature range of 33 to 82 ° C.

For biological control agents the application of the drying processes is not obvious, as, as previously mentioned, it was not convenient to apply various drying processes, including one of low thermal aggression such as in the case of lyophilization, to obtain high yields of viable cell recovery, which in turn are effective against pathogens (Abadias et al., 2001; Jones et al., 2004; Abadias et al., 2005). Therefore, the processes object of the present invention constitute a novelty. Drying by lyophilization is very expensive and, as mentioned earlier, no good results were obtained with its application to yeasts used as biological control agents. Fluid bed drying has been widely studied and used to produce baking yeast, requires equipment at a lower cost than that required for lyophilization, but is more expensive than that used for spray drying, and requires longer times. retention and greater care in the relative humidity of the drying air. Finally, Silva et al. (2002) report that spray drying can be used to dry large amounts of biomass at a relatively low cost. Powders dried in this manner can be transported at low cost and stored stably for extended periods of time. On the other hand, Janisiewicz and Jeffers (1997) indicate that the major obstacle in the commercialization of products of biological control agents is the development of products formulated to have a stable shelf life, conserving an efficiency similar to that presented by fresh cells of the same. agent. Therefore, spray spray drying offers the possibility of having large quantities of products formulated with biological control agents, with long shelf life and effective for application in commercial orchards in the field. Spray drying of R. minuta, harvested at a stationary phase, allowed to obtain solid compositions with a shelf life conveniently greater than previously developed liquid formulations (Patino-Vera et al., 2005) (see example 3). . With shelf life under refrigeration of up to one year, for excellent later use as a biological control agent for C. gloeosporioides, the causative agent of mango anthracnose. This long shelf life may be due to the fact that concentrating and drying the cells noticeably reduces water activity (aw) and some toxic substances for R. minuta such as hydrogen peroxide and carbon dioxide are decomposed or evaporated. formaldehyde (compounds derived from yeast metabolic activity). On the other hand, R. minuta solid and dry compositions obtained by the method of the present invention were not affected in their biological control ability of mango anthracnose, unlike in the other previously reported yeast drying cases.

Following the method of the present invention, it is possible to produce a dry solid composition effective in the biological control of C. gloeosporioides and having a shelf life under refrigeration of at least one year, characterized by the fact that it comprises viable R. minuta cells.

Moisture levels of dry yeasts below 5% (w / w) are not recommended because cells can suffer irreversible biochemical damage (Masters, 1985) and with the drying procedure proposed in the present invention it is possible to obtain solid, dry compositions. characterized by the fact that they comprise R. minuta cells, with a minimum moisture content of 5.23 ± 0.59% and a maximum of 7.68 ± 0.59% (see example 2).

In the practical application of biological control agents, it is normal to use doses with high concentrations of viable cells (approximately 107 to 10 CFU / mL) (Janisiewicz and Korsten, 2002), therefore, it is necessary that commercial presentations of compositions with biological control agents have at least an order of magnitude greater in viable cell concentrations to transport the smallest possible amount of solvent and thereby minimize the costs of transport, storage and handling of the composition. In the present invention a dry and effective solid composition has been developed with a viable cell concentration of at least 1 x 10 9 CFU / g yeast R. minuta. The minimum shelf life recommended for compositions with biological control agents is six months (Janisiewicz and Korsten, 2002), to be compatible with routine management and storage practices in the agrochemical market. The dry and biologically effective solid composition of C. gloeosporioides has a shelf-life under refrigeration of at least one year and is characterized by the fact that it comprises viable R. minuta cells.

As mentioned earlier, several authors have reported inconsistent efficacy of biological control agents when they are applied to commercial orchards (Janisiewicz and Jeffers, 1997; Janisiewicz and Korsten, 2002; Guetsky et al., 2001) and the benefits to be achieved. can achieve by employing mixtures of biological control agents (Janiciewicz and Jeffers, 1997; Janisiewicz and Karsten, 2002; Spadaro and Gullino, 2004). For this reason, the present invention also comprises a composition comprising a second biological control agent and requiring a lower dose of each biological control agent than that in independent application thereof to achieve similar levels of biological control. As reported by the authors of the present invention (Carrillo-Fasio et al., 2005) Bacillus subtilis was the biological control agent with the suitable characteristics to be mixed with R. minuta, therefore, the solid and dry composition of the present invention, which comprises R. minuta, may be mixed with solid and dry B. subtilis compositions, which may be premixed or packaged and prepared for mixing and resuspension prior to application.

This type of solid and dry composition of R. minuta produced in semi-commercial quantities was applied to mango orchards in Sinaloa State, Mexico mixed with Bacillus subtilis (an effective biological control agent against multiple fungal diseases). To obtain the required amounts of Bacillus subtilis, spores of this microorganism were produced as reported by Carrillo-Fasio et al. (2005).

R. minuta compositions showed better control of anthracnose severity compared to the commercial chemical fungicide Benomilo (trade name: Benlate), the same in liquid compositions with R. minuta as the sole biological control agent, as when contained. additionally B. subtilis in liquid or solid compositions. Applications of liquid or solid R. minuta + B. subtilis compositions used in commercial cv Kent mango orchards were able to reduce the severity of anthracnose by up to 86.7% compared with the fruits of a controlled treatment (in only irrigation water was applied) after 15 days of storage (see examples 4 and 6). An additional advantage of using the antagonist mixture is that it allows the necessary viable microorganisms to be reduced by up to two orders of magnitude over the cases in which the antagonists were used separately. The use of biological control agents allows the possibility of exporting agricultural products to countries of the European Economic Community and others such as Japan, where the value of the fruit is even higher than that quoted in the United States.

This is a substantial improvement over the state of the art due to the fact that no reports were found where R. minuta yeast is employed in solid composition, nor mixed with liquid or solid formulations of B. subtilis, for the control of fungal diseases. plants of commercial interest, particularly fruit.

Accordingly, a method for the biological control of Colletotrichum gloeosporioid.es is claimed which comprises at least one pre-harvest application of effective doses of a dry and effective solid composition comprising the biological control agent Rhodotorula minuta.

Ideally, the method for biological control of C. gloeosporioides, which is comprised in the present invention, implies that the composition is spray applied to the entire aerial part of the plant to be treated.

As can be deduced from the present invention, the solid and dry compositions of R. minuta, that is, only with this yeast or applied in a mixture with Bacillus subtilis, have wide field utility for their effectiveness in controlling anthracnose of mango. caused by C. gloeosporioides, without detriment to fruit quality, because other fruit quality indices such as acidity, pH and total soluble solids are not affected (see example 5), including improving their shelf life, losing a smaller amount of water during their postharvest storage makes them suitable. The results of the method for biological control of C. gloeosporioides are as good or superior to those obtained by chemical treatment (see examples 4 and 6), and are therefore suitable for their handling in the agrochemical market.

Although C. gloeosporioides is the causal agent of anthracnose in tropical and subtropical crops such as: mango fruits (Mangifera indica), papaya (Carica papaya), avocado (Persea americana), mandarin (Annona muricata), tangerine (Citrus reticulata, C. unshiu and C. reshni) and rough lemon (Citrus jambhiri Lush) (Arauz, 2000; Gamagae et al., 2004; Freeman et al., 1998; Alvarez et al., 2004; Contreras y Rondón, 1985); smaller fruits such as strawberry (Fragaria ananassa L.) (Freeman et al., 1998); plants whose interest is their flowers, such as orchids (Orchidaceae); it even affects tuber crops such as yam (Dioscorea sp.) (Perez-castro et al., 2003); among others, in the present invention the case of mango is employed to demonstrate the field effectiveness of solid and dry compositions comprising R. minuta, without implying that it only works with mango as a white crop. Said compositions are effective in the biological control of C. gloeosporioides, and are therefore useful in the biological control of diseases caused by C. gloeosporioides in mango or any other crop.

MATERIALS AND METHODS

Strain of the pathogen Colletotrichum gloeosporioides, the phytopathogen mushroom was isolated from anthracnose mango fruits and identified according to their morphological characteristics (Freeman et al., 1998). The strain is maintained on potato dextrose agar (PDA) medium (BD Bioxon, Mexico) under refrigeration at 4 ° C.

A strain of the Rhodotorula minuta yeast biological control agent, this yeast forms spherical, domed, bright pink colonies with smooth edges and is unable to form spores or mycelium in NYDA solid culture medium (8 g / L, yeast extract 5 g / l, dextrose 10 g / l and 18 g / l agar). She belongs to the Deuteromycetes; Order Cryptococcales; Criptococcaceae family; Rhodotoruloidea subfamily, genus Rhodotorula (Girard and Rougieux, 1964). The Rhodotorula minuta strain was kept under refrigeration conditions at 4 ° C in slanted tubes with potato dextrose agar (PDA) medium (BD Bioxon, Mexico). Since mango was used in the present invention to illustrate field application, the target plant's yeast was isolated (as is common in the biological control area) by following the procedure reported by the authors of the present invention. in Patino-Vera et al. (2005). Similarly, R. minuta strains can be isolated from the philosopher of other crops for which C. gloeosporioides is pathogenic. However, strains are also available from the ATCC, with 13 records in its Fungi, Yeasts & Genetic Stock catalog, for example: strains numbered 10658, 14926, 16731, 16732, 16733, 16741, 208876, 2776, 32769 and 36236. .

Cultivation media for R. minuta. Inoculum and Seed Medium (PYD) (Gil): potato starch 4.0, dextrose 20.0 and yeast extract 5.0; Production Medium (Enriched Mineral Medium) (g / l): dextrose 34.4, yeast extract 5.0, (NH4) 2HP04, 2.787 KH2P04, 2.05 MgSO4 * 7H20, 0.1 NaCl, 0.009 FeSO4 * 7H2 O, 0.055 CaCl2, 0.01 CuSO4 * 5H2 O and 0.0076 MnSO4 * H2 O.

Strain of bacterial biological control agent Bacillus subtilis. The Bacillus subtilis strain is characterized by having colonies of rhizoid morphology, wavy edge, granular surface and mucoid consistency (when aged they become dry and brittle) when grown in a solid medium. The color of the colonies is bright creamy white (in young colonies) and opaque cream (as they age). In staining Gram's differential dyeing is positive, with the shape of flagellate bacilli. To reactivate the strain, it is reseeded in a glycerol tube in a Petri dish with sterile YPG medium (yeast extract 10 g / L, peptone 10 g / L and dextrose 10 g / L; 15 g / L of agar) and subsequently incubated at 30 ° C for 24 hours. Since mango was used in the present invention to illustrate field application, the target plant phyllosphere bacteria (as is common in the biological control area) were isolated following the procedure reported by the authors of the present invention. in Carrillo-Fasio et al. (2005). Similarly, strains of B. subtilis can be isolated from the phyllosphere of other crops for which C. gloeosporioides is pathogenic. However, there are also strains available from the ATCC with more than 200 records including 31578, 33677, 35148, 39085, 39086, 39087, etc.

Cultivation media for B. subtilís. Inoculum and Seed Medium (YPG) (g / L): yeast extract 10, peptone 10 and dextrose 10; Production medium (g / L): 4.00 (NH 4) 2 SO 4, 5.32 K 2 HPO 4, 6,40 KH 2 HPO 4, 0,4 MgSO 4 * 7H 2 O, 0.005 MnCl 2, 0,040 CaCl 2, 0,030 FeSO4 * 7H20 and dextrose 10.0.

Phosphate buffer solution (for liquid formulation) (g / L): sodium chloride 8.0, monobasic potassium phosphate 2.0, potassium chloride 2.0 and dibasic sodium phosphate 2.9.

Viable cell count. Determined manually by the plate count method, quantifying Colony Forming Units (CFU).

Determination of reducing sugars. To determine glucose concentration, 1 mL of the fermentation broth is taken, which is centrifuged for three minutes at 13,000 rpm in an Eppendorf centrifuge; from the supernatant, dextrose is determined with a Yellow Spring Scientific Instruments (YSI) Model 2700 enzyme analyzer. This analyzer employs an enzyme (oxidized glucose) that carries out a catalytic reaction that produces hydrogen peroxide, which , when oxidized on a platinum anode, produces an electrical signal which is directly proportional to the dissolved glucose concentration in the sample.

Determination of shelf life of liquid formulations. Once liquid formulations are obtained, they are stored under refrigeration and evaluated periodically by taking 1 mL of liquid formulation and applying the viable cell count (CFU) procedure over the period of time to be monitored.

Determination of shelf life of solid formulation. Once the different yeast suspensions have been dried, they are stored in a cold room at 4 ° C and viability is assessed each month. The procedure consists of weighing 100 mg of powder, resuspending in a test tube with 10 mL of 0.85% sterile saline (using NaCl), perfect homogenization (to rehydrate cells) and making the corresponding dilutions. , to determine CFU / g by the same viable cell count procedure.

Determination of moisture content of solid formulations. 100 mg of solid formulation is weighed in triplicate on previously tared aluminum scales; They are then placed to dry in an oven at 105 ° C for two hours and then stored in a desiccator for cooling and constant weight. Moisture is obtained by weight difference before and after drying.

Determination of water activity, the water activity of the samples is determined using an electric hygrometer, brand name "Novasina AVJ Sprint", model TH500, according to the manufacturer's instructions.

Field treatment application. To corroborate field effectiveness, a commercial mango cv. Eight-year-old Kent, located in Rosario, Sinaloa, Mexico. The trees were homogeneously selected to be distributed for each treatment (three trees per treatment). In each treatment a monthly application (five in total) was performed using a back-loading sprinkler pump operated at 400 pounds of pressure. It was started from flowering (February) and until harvest (June or July) by spraying fresh or dried (previously hydrated) cell suspensions produced in pilot fermenters. 7L of the cell suspension was sprayed onto each selected mango tree until all the foliage was moistened. As a control treatment, 7 L of water was sprayed to water.

Quantification of anthracnose severity. From each treatment, 100 mango fruits were harvested at physiological maturity (Báez et al., 1993), which were stored under market simulation conditions at 20 ° C and 85% relative humidity. The severity of the disease was evaluated after a minimum of fifteen days after storage, using as an indicator the presence of disease symptoms (anthracnose) in the fruits. For this, an adaptation of the hedonic scale proposed by Smoot and Segall (1963) was used, where: 0 = healthy; 1 = strokes (chlorotic spots); 2 = slight (dark lesions 1-5 mm in diameter); 3 = medium (dark lesions larger than 6 mm in diameter), · and 4 = severe (dark sunken lesions with fungal structures).

Evaluation on fruit quality. Weight loss was assessed every third day by weight difference with respect to initial fruit weight, expressed as a percentage (Díaz-Pérez, 1998). The pH, titratable acidity (TA) and total soluble solids (TSS) were quantified according to the methodology proposed by AOAC (1998) at 0, 6 and 12 days of storage. For the determination of pH and acidity a Mettler automatic titrator (model DL-21) was used and the results were expressed in units of pH and citric acid percentage, respectively. Total soluble solids were determined with a temperature compensated Abbe Leica Mark II refractometer and previously calibrated with pure water. Results were expressed in degrees Brix (° Brix). For quality parameters, one fruit was selected as the experimental unit and five replications were used.

Statistical analysis. The study of the severity of the disease in mango fruits was analyzed using a completely randomized block design. The data obtained from the evaluations of each treatment were submitted to Friedman's unilateral variance analysis (p <0.05) using the SAS statistical system (SAS, 1998). The difference between treatments was determined by Tukey test (p = 0.05). For the differences in the variance analysis (ANOVA) of the quality variables, the means were separated by Tukey's multiple comparison test with a 5% error probability, using the Minitab version 13.1 statistical program.

EXAMPLES

In the following examples the present invention is best illustrated, but certainly without restricting its scope. EXAMPLE 1. Production of stationary phase R. minuta cells in a stirred 10 L fermenter.

It began with the propagation of the Rhodotorula minuta strain in Petri dishes with PDA medium by transferring the yeast from a previously inoculated and incubated slanted tube with abundant yeast growth using a microbiological seed tray following the usual microbiological techniques for ensure sterility and avoid contamination of materials and culture media. Petri dishes were streaked and incubated at 29 ± 1 ° C for 24 to 48 hours. When notorious yeast growth was achieved throughout the stria, three trays were transferred per 250 mL Erlenmeyer flask with 25 mL of PYD medium and incubated at 200 rpm shaking at 29 ± 1 ° C, for 24 hours. At least two 250 mL test flasks were prepared to transfer 50 mL of these pre-inocula to a 2.8 L wide-bottom or Fernbach test flask with 450 mL of sterile PYD medium for subsequent incubation with a stirring at 20 rpm at 29 ± 1 ° C for 24 hours. At least two Fernbach test flasks were prepared in order to transfer one liter of this inoculum to a 14 L nominal fermentation jar containing 10 L of Enriched Mineral Medium (mem). The jar was equipped with three Rushton 6 flat vane turbines and a point diffuser. Stirring was performed at 240 rpm at 29 ± 1 ° C with aeration of 10 1 / min for 48 hours. In order to know the cultivation time in which R. minuta yeast is in a stationary phase, its growth and glucose consumption kinetics (the main carbon source of the production medium) was determined. As shown in figure 1, it is possible to recognize when the yeast is in a stationary phase by measuring glucose consumption. When glucose is in a concentration range between 0 and 2 g / L (between 35 and 48 hours of cultivation), it is evident that R. minuta growth was arrested and kept at virtually the same concentration of CFU / mL; therefore, it is in a stationary phase, at which stage it is possible to successfully dry this yeast. EXAMPLE 2. Formulation and spray drying of Rhodotorula minuta.

In order to develop the formulation and drying process of a solid composition effective in the biological control of mango anthracnose, with Rhodotorula minuta, two steps were taken. The first condition was to obtain the cell mass by centrifuging the broth harvested at the end of the fermentation process (as described in example 1) using a MiniSharples (Type CL-I-1) tubular centrifuge with a bowl diameter of 1 , 75 inches in an operating range of 8,000 to 12,000 rpm. Thereafter, said mass was resuspended in phosphate buffer such that the suspension had a concentration of 6% total solids. In the second condition, the dryer was fed with the complete broth as harvested from the fermenter, which contains a total solids concentration of 3%. All drying experiments were performed under the same operating conditions: 120 ° C inlet temperature, 50-52% drying air relative humidity (HR), 180 mL / min liquid suspension feed flow 50-60 ° C outlet temperature, a total solids concentration of approximately 3 or 6% and from stationary phase harvested R. minuta biomass (see example 1). The results corresponding to this example are shown in table 1 and figure 2.

Table 1. Summary of drying results of solid formulations As indicated in Table 1 and Figure 2, the spray drying process affected the proportion of viable R. minuta cells recovered because of the centrifuged broth they were recovered on average 23.9. ± 8.6% viable cells; while from the complete broth 19.6 ± 8.3% of them were recovered. Regarding the solids recovery efficiency, the lowest values were obtained when the broth is centrifuged, due to the fact that it contained a smaller amount of dissolved solids (such as mineral salts), which were eliminated with the previous process. centrifugation.

On the other hand, the viable cell retrieval efficiency (CFU) is slightly lower when the whole broth is fed (without centrifugation) compared to the case of the resuspended cell package. However, the concentration of viable cells per gram (measured as CFU / g) is still found in the order of 1010 and the solids recovery efficiency is higher. Therefore, there are two process variants for obtaining solid formulations of R. minuta. The first is to feed the spray dryer with the full harvested broth (without using the centrifuge operation), which requires less equipment investment and simplifies the process, reducing operating costs. The second process is performed by centrifugation and resuspension of R. minuta cell mass in phosphate buffer, which gives a greater number of living cells per gram (2.3 times greater than without centrifugation) in the product. dry recovered from the dryer. This should allow the use of a significantly smaller amount of product to be applied to the same number of trees. The latter composition is characterized by the fact that by centrifuging and discarding the supernatant, the greatest amount of residual soluble solids from R. minuta broth is eliminated.

As can be seen from the data given in Table 1, the water activity (aw) of the solid compositions obtained by the method of the present invention is relatively low. This contributes to yeast cells remaining with very low metabolic activity, since most microorganisms are unable to thrive in very low aw environments, so they die or dehydrate, or pass into latent conditions during an indefinite time (Madigan et al., 1999; Paul et al., 1993).

On the other hand, the moisture content of solid formulations is in the appropriate range as it has been reported that moisture contents between 5 and 8% do not cause irreversible damage to metabolic functions of microbial cells (Masters, 1985).

To prove that the solid and dry composition with R. minuta, object of the present invention, preserves the biological control capacity of the Colletotrichum gloeosporioides mushroom, pathogen causing anthracnose in mango, an in vitro bioassay was performed using 24-well microplates. , similar to what was reported by Janisiewicz et al. , (2000). Through this bioassay, the curves of the percentage of phytopathogen germination were obtained. As can be seen in figure 3, during the 50 hours of cultivation, the number of conidia remained virtually constant for both cases, allowing time for them to germinate approximately 30% of them (in the control) and less than 10% in the bioassay. dry solid composition with R. minuta.

In Figure 3, comparing the control experiment (without the dry solid composition of R. minuta) and the one with yeast, the effect of inhibition of conidial germination of the phytopathogen C. gloeosporioides mushroom exerted by the dry solid composition of C. gloeosporioides can be clearly observed. R. minuta. The difference between the two conditions was up to 25%, whereby the solid and dry composition obtained by the method of the present invention object of the present invention can be said to have retained virtually the same antagonistic effect in in vitro assays. than a liquid composition with fresh cells of the same biological control agent.

On the other hand, it was also corroborated by the fact that yeasts of solid and dry composition present a degree of antagonism similar or equal to that of liquid formulation, previously reported by our research group (Patino-Vera et al., 2005). Percent germination results at 56 hours of cultivation are shown in Figure 4. This figure shows that the dry and solid composition had a percentage germination inhibition effect virtually equal (without significant difference) to the net composition at the same time. R. minuta dose of 108 CFU / mL. EXAMPLE 3. Shelf life of developed solid formulations containing R. minuta as compared to that of liquid formulations.

In order to compare the shelf life of the two types of formulations, the viable cell concentration of Rhodotcrula minuta was monitored in both liquid and solid compositions developed and which are the subject of the present invention.

Fresh liquid compositions were obtained as reported by Patino-Vera et al. (2005). After the fermentation process, the cell package (obtained by centrifugation in a Minisharples team) was resuspended in phosphate buffer with a ratio of 0.333 mL buffer / g wet mass. The shelf life of the solid and liquid formulations was obtained by storing both formulations at 4 ° C and, as can be seen in figure 5, the concentration of viable cells in the solid composition object of the present invention is maintained at levels of 1010 ° C. CFU / g for a significantly longer period of time (ten months or more) than for liquid composition (in phosphate buffer). The liquid formulation loses almost 90% of its viable cell concentration in the first month, while solid formulation concentration only drops 5% in the same period. In the second month, the concentration of viable Rhodotorula minuta cells in the liquid formulation decreases by more than two orders of magnitude, while the solid and dry composition still has 80% of the viable initial population. The above may be due to the fact that in the drying process a large part of the water content and low molecular weight volatile toxic substances are eliminated, as well as having diffusional barriers (by formation of gels) and limiting cellular metabolism, because Dehydration caused by drying has been reported to decrease the availability of water within cells, so cells enter a latent state in which metabolism is almost completely disrupted (Paul et al., 1993).

The developed solid and dry compositions which are described in the present invention maintain a high concentration of viable cells (= 1010 CFU / g) for up to twelve months of storage (Figure 5). This period of time is sufficient and acceptable for the purpose that a biological control product may be marketed (Pusey, 1994). EXAMPLE 4. Field trials with R. minuta liquid compositions as the sole biological control agent and with a second biological control agent, B. subtilis, which illustrates the efficacy of these microorganisms as biological control agents against Colletotrichum gloeosporioides.

Field applications were pre-harvested following the procedures described in Materials and Methods. The doses and formulations used are shown in table 2.

Table 2. Treatments and dose applied to a mango orchard (Mangifera indica) cv. Kent to control anthracnose (caused by C. gloeosporioides).

Accumulated anthracnose severity data (expressed as% of severity) at 15 days after fruit storage are shown in Table 3. Values of up to 60.5 disease severity were obtained for the absolute control fruits and 41 7 for chemical treatment. The results indicated (see table 3) that, with the combined application of antagonists, greater control of anthracnose (lower severity of disease, up to only 8.0 in terms of severity) is obtained, with significant differences between treatments being found. applied and showing drastically lower values of gravity with respect to chemical application and absolute control. An important advantage of using the composition with R. minuta as the first biological control agent and B. subtilis as the second object of the present invention is that it allowed the highest anthracnose control levels to be obtained using concentrations of two orders of magnitude less than for the separate application of antagonist microorganisms (from 108 to 106 CFU / mL for yeast and from 106 to 104 CFU / mL for bacteria, see Table 2); This makes this treatment more attractive for its commercial application, since 100 times fewer cells of each of these biological control agents should be applied and still achieve better anthracnose control. Reducing the severity of anthracnose in treatments with antagonist mixtures indicates a possible synergistic effect between yeast and bacteria. Application of mixtures of antagonist microorganisms reduced variability and improved biological control effectiveness in many systems that include fruit pathogens (Guetsky et al., 2001). However, this is the first time it has been reported for the control of anthracnose caused by C. gloeosporioid.es, proven at a semi-commercial level, using mango as a white crop. In the present example, Kent mango compositions clearly resulted in greater efficacy than chemical treatment, possibly because antagonists colonized the surface of leaves and fruits and thus minimized premature or latent infections of mango fruits.

Table 3. Severity range of anthracnose in mango fruits (Mangifera indica) cv. Kent treated pre-harvest. * Values with different letters have a statistically significant difference (Tukey, p <0.05). EXAMPLE 5. Effect of application of the developed biological control agent compositions on the quality of the fruits produced.

The same compositions presented in Table 2 were used to study the effect of application of the biological control agents, object of the present invention, on the quality of mango cv. Kent

One of the most important parameters for assessing the quality of mango fruits is weight loss during storage. When analyzing the behavior of this variable, it was observed (see table 4) that, at the beginning of the market stage (storage day 3), all fruits showed a similar behavior with a weight loss of less than 1%. It is from the ninth day of storage at 20 ° C, when statistical differences in weight loss with respect to control (p <0.05) were observed. On the fifteenth day, values of 4.6 and 4.3% of weight loss were presented for control and chemical fruits, respectively, and less than 3.6% for fruits of the treatment with yeast composition ( R. minuta 108 CFU / mL).

Table 4 Changes in weight loss of mango fruits (Mangifera indica) cv. Kent, during storage for up to fifteen days at 20 ° C and 85% relative humidity, treated with biological antagonists, chemical fungicides and absolute control. * 0s Values with the same letter between columns are statistically equal (Tukey, p <0.05).

Other quality parameters evaluated were pH, acidity and total soluble solids content in the fruit. By analyzing these, it was observed that the treatment with both compositions (R. minuta and R. minuta + B. subtilis) had the lowest pH values, being statistically different from the control fruits at the beginning of storage (Table 5). . On the other hand, no relationship between pH and results on citric acid content was observed. The pH tended to increase during fruit ripening, associated with a considerable drop in titratable acidity. At twelve days of storage, the treatment of the B. subtilis and R. minuta mixture had the highest pH value. The percentage of citric acid started at high (and statistically equal) values and was significantly reduced after twelve days of storage with B. subtilis and R. minuta + B. subtilis mixtures up to 0.06 acidity. and 0.04, respectively, where the highest value was presented by the absolute control (0.14). The greatest reduction in acidity of fruits treated with biological antagonists is probably due to the fact that microorganisms use organic acids to perform respiratory activity (Ruiz and Guadarrama, 1992). Similarly to pH, the total soluble solids content was not affected by the application of the different treatments (Table 5), which showed - on average - an increase of 8 to 14 ° Brix during the first twelve days of storage. According to Seymour et al. (1990), the increase in total soluble solids is due to the degradation of macromolecules to simpler sugar molecules, reporting to Kent mango a change in total soluble solids from 7.7 to 16.1. ° Brix during maturation for twelve days at 20 ° C.

Therefore, the application of R. minuta and B. subtilis compositions did not negatively affect the main quality parameters of mango cv Kent. On the contrary, there is less weight loss during storage and since this product is traded per unit weight (tonnes), producers would have more profits in its marketing.

Table 5. Changes in pH, titratable acidity and total soluble solids of mango fruits (Mangifera indica) cv. Kent during storage for twelve days at 20 ° C and 85% relative humidity, treated with biological antagonists, chemical fungicide and absolute control. * The values with the same letter within each column are statistically equal (Tukey, p <0.05). EXAMPLE 6. Field trials with dry solid R. minuta compositions as the first biological control agent and a second biological control agent, B. subtilis.

Field applications were pre-harvested following the procedures described in Materials and Methods. The doses and formulations used are presented in table 6.

Table 6. Treatments and dose applied to a mango orchard (Mangifera indica) cv. Kent to control anthracnose (caused by C. gloeosporioides).

Accumulated anthracnose severity data (percent severity) at the fortnight after fruit storage can be seen in Table 7, and in this table it is appreciated that there is a statistical difference between treatments (P <0.05). The treatment (R. minuta 106 CFU mL'1 + B. subtilis 104 CFU mL "1) showed greater efficacy in the control of anthracnose in mango fruits than Benomilo and the absolute control. The respective percentage of severity for this biological treatment. 23.1%, followed by chemical treatment (36.2%) and absolute control (46.8%), an important advantage of using a composition with R. minuta as the first biological control agent and B. subtilis as a second object of the present invention is that it has enabled higher levels of anthracnose control to be achieved using concentrations two orders of magnitude lower than in the case of separate application of antagonist microorganisms (from 108 to 106 CFU / mL in the case of yeast and 10e to 104 CFU / mL for the bacterium, see example 4), this confirms that treatment with the mixture is more attractive for its commercial application as 100% fewer cells would have to be applied. each of these acts biological control and yet obtain better anthracnose control than chemical fungicide. As mentioned earlier in the present example, the solid and dry compositions that are comprised in the present invention used in Kent mangoes clearly resulted in greater effectiveness than chemical treatment, possibly because antagonists colonized the leaf and fruit surface and thus minimized premature or latent mango fruit infections.

Table 7. Severity range of anthracnose in mango fruits (Mangifera indica) cv. Kent treated pre-harvest. * Values with different letters have a statistically significant difference (Tukey, p <0.05). EXAMPLE 7. Effect of formulation by incorporating supports or thermoprotectants on the production of dry solid composition with R. minuta.

Through the fermentation process previously described, 20 L of Rhodotorula minuta broth were obtained. From these, 20 mL samples were taken to determine the total solids content (TS) in duplicate, obtaining an average of 3.4% in the freshly harvested broth. Subsequently, the amount of support needed to have a total solids concentration of 10% was added and the spray drying aliquots of 4385 mL were taken. Corn starch (F. maiz) was used as support in the following composition: ST of R. minuta broth 3.4% + 6.6% of corn starch solids.

Once the above suspension was prepared, it was subjected to a spray drying process under the following drying conditions: inlet temperature 120 ° C, feed flow rate of 106 mL min -1, dryer outlet temperature in 60 - 65 ° C and 50 to 52% RH of the drying air.

The viability (9.43 x 109 CFU / g), the percentage of recovered solids (87.5) and the residual moisture (7.21%) of the dry powder were determined. To estimate the effect of the addition of a support on shelf life, some of the compositions object of the present invention have been subjected to an accelerated aging process. For this, a triplicate 3 g sample of the dry formulation was taken and placed in an oven at 37 ° C where it was kept for a storage period of approximately 65 days. Periodically, 0.1 g samples of the powder stored at different time intervals (at 5, 15, 25, 45 and 65 days) were taken, making a viable cell count (measured as CFU) by plaque counting. Shelf life data of the two formulations developed are shown in Figure 6. Use of the support allowed to maintain a viable cell concentration (measured as CFU) of 1 x 109 CFU * g_1 at a temperature of 37 ° C for time periods. much longer (65 days storage) compared to unsupported formulations; which at 24 days no longer had viable cells (measured as CFU).

As mentioned earlier, in addition to being the first to obtain solid and dry R. minuta compositions as a biological control agent by obtaining a high concentration of viable cells (CFU measures) and high efficacy in the control of fungal diseases, we are also the only ones to. have dry compositions with a high concentration of viable cells (approximately 1 x 10 9 CFU gx) that can be stored at 37 ° C for up to 65 days. This is very important due to the fact that fruits such as mango, papaya, avocado, strawberry, yam, etc., are produced in tropical or semitropical zones, with average temperatures around 37 ° C, allowing a better commercialization of the compositions object of the present invention, since, if necessary, their exposure up to 65 days at room temperature (of 37 ° C on average) should not significantly affect their viability, thus presenting a technical advantage. -Additional commercial. As previously reported, several authors have failed to develop dry products (Wan-Yin and Mark, 1995; Jones et al., 2004), including other yeasts used as biological control agents that retain their efficacy in controlling fungal diseases, even under cold storage (Abadias et al., 2005).

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All publications, patents and applications cited herein are incorporated by reference in their entirety in the description.

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Having described the present invention, it is considered to be novel and therefore the object of the following claims is claimed as property.

Claims (19)

1. SOLID, DRY COMPOSITION, EFFICIENT IN BIOLOGICAL CONTROL OF COLLETOTRICHUM GLOEOSPORIOIDES, having a shelf life under refrigeration of at least one year, comprising at least 1 x 109 CFU / g of steady-state Rhodotorula minuta, a pH buffer neutral and a protective stand. SOLID COMPOSITION according to claim 1, characterized in that the buffer is phosphates. SOLID COMPOSITION according to claim 1, characterized in that the protective support is maize starch. SOLID COMPOSITION according to claim 1, characterized in that it comprises a moisture content between 4.6 and 8.4%. Method for producing a composition as defined in claim 1, comprising the steps of: a. cultivar submerged fermentation Rhodotorula minuta (R. minuta) in an enriched mineral medium, with the following values for the control parameters: temperature from 20 to 30 ° C; agitation from 100 to 400 rpm; aeration of 0.5 to 1.0 volumes of air per volume of medium per minute (WM) and pH of 4 to 9; for 40 to 65 hours, sufficient for the cultivation to reach the stationary phase; B. recovering R. minuta cells by centrifugation and resuspending them in a phosphate buffer; ç. formulate by the addition of a high starch flour-based thermoprotectant to the cell suspension; d. drying the cell suspension at a temperature between 50 and 160 ° C to preserve a convenient proportion of viable cells in the resulting composition. Method according to Claim 5, characterized in that the high starch flour for its formulation and drying is selected from the group consisting of maltodextrin, maize flour and cornstarch. Method according to Claim 5, characterized in that the drying is carried out in a spray dryer at an inlet temperature between 100 and 160 ° C, and an outlet temperature between 50 and 70 ° C. Method according to claim 5, characterized in that the cell suspension has R. minuta cell concentration between 1 and 6%, preferably 3.4% on a dry basis. METHOD FOR BIOLOGICAL CONTROL OF NURSES CAUSED BY COLLETOTRICHUM GLOEOSPORIOIDS, characterized in that it comprises at least one pre-harvest application of effective doses of the composition as defined in claim 1. Method according to claim 9, characterized in that it is sprayed onto the entire aerial part of the plant. Method according to Claim 9, characterized in that the anthracnose is controlled to levels equal to or greater than those obtained by treatment with a chemical fungicide. Method according to Claim 9, characterized in that the crop to be treated is a tropical or subtropical crop. Method according to Claim 9, characterized in that the crop is selected from the group consisting of mango (Mangifera indica), papaya (Carica papaya L.), avocado (Persea americana), mandarin orange (Annona muricata), mandarin (Citrus reticulata, C. unshiu and C. reshni), rough lemon (Citrus jambhiri Lush), strawberry (Fragaria ananassa L.), orchids (Orchidaceae) and yam (Dioscorea sp.) - Method according to claim 13, characterized in that the crop to be treated is mango (Mangifera indica). Method according to claim 14, characterized in that the mango crop to be treated is of c.v. Kent Method according to claim 9, characterized in that it comprises at least one pre-harvest application of a mixture of effective doses of a composition as defined in claim 1 and Bacillus subtilis. Method according to claim 16, characterized in that the effective pains are at least 1 x 10 6 CFU / ml R. minuta and 1 x 10 4 CFU / ml Bacillus subtilis. Method according to Claim 17, characterized in that the combined action of the combination of R. minuta and Bacillus subtilis is synergistic. METHOD FOR REDUCING WEIGHT LOSS DURING MANGO STORAGE, characterized by pre-harvesting at least one application of a composition as defined in claim 1.